Cultivation and Genetic Manipulation of Free-Living and Pathogenic Leptospires

钩端螺旋体病是由钩端螺旋体属成员引起的全球性人畜共患疾病。钩端螺旋体病虽然传播广泛，有时甚至致命，但被认为是一种被忽视和研究不足的疾病。钩端螺旋体病的病原体于1916年首次被发现，但体外生长速度缓慢和用于操纵这种螺旋体基因组的遗传工具有限，阻碍了对毒力因素的确定和疫苗的开发。

Leptospirosis is a global, zoonotic disease caused by members of the genus Leptospira. Although widespread and sometimes fatal, leptospirosis is considered a neglected and understudied disease. The causative agent of Leptospirosis was first identified in 1916 but the slow in vitro growth rate and limited genetic tools with which to manipulate the genome of this spirochete have hampered the identification of virulence factors and development of a vaccine. Leptospires can be broadly divided into two groups: free-living saprophytes and infectious pathogens. The most widely used and studied species are L. biflexa (a non-pathogenic saprophyte) and L. interrogans (a pathogen). However, the non-pathogenic L. biflexa is more easily cultivated and more amenable to genetic manipulation than the pathogenic L. interrogans. Therefore, we have focused on L. biflexa to master the microbial and genetic techniques needed to manipulate this genus, with the intention to transfer this expertise to the more refractory pathogenic strains. Targeted gene inactivation, shuttle vector transformation, and transposon mutagenesis have all been successfully used in L. biflexa. To date, no shuttle vector system exists for pathogenic species and there are few published reports of targeted gene inactivation in L. interrogans. Transposon mutagenesis can be applied to L. interrogans but it functions at such a low efficiency that it cannot be utilized for any broad applications, such as auxotrophic screens or signature tagged mutagenesis. The lack of a shuttle vector for L. interrogans hinders complementation and thus limits interpretation of any resulting phenotypes of transposon or targeted deletion mutants. Since L. biflexa has a better transformation frequency than other species we plan to optimize new techniques in this organism. In FY2014 we have begun to evaluate different systems that may affect the transformation effiencies of leptospires. The lamda red recombinase system has been used successfully in other bacteria to improve targeted mutagenesis. We have begun to assess this system in L. biflexa, and if it appears promising, we will test it in the pathogen L. interrogans. Also, we are studying the CRISPR/Cas system that is present in L. interrogans but absent in L. biflexa. This system targets and degrades foreign DNA and we hypothesize that it may contribute to the lower transformation frequency observed in the pathogen relative to the saprophyte. Specifically, we have demonstrated that the CRISPR/cas operon is transcribed during in vitro growth and have integrated part of the operon into L. biflexa and have shown that the genes are also transcribed in this heterologous host. Currently, we are attempting to inactivate specific cas genes in L. interrogans and move the entire operon into L. biflexa. We proceeded in FY2014 to develop a proteomic map of in vitro cultivated L. biflexa to identify highly expressed proteins from membrane- and soluble-fractions. We have identified abundantly-expressed proteins that can be used as cellular markers, as controls for gene expression studies, and also quantified the transcript data from a subset of these genes. Further, we demonstrated that a significant number of L. biflexa proteins are subject to post-translational modification including phosphorylation and acetylation. Highly expressed proteins allow us to identify targets that may play important physiological roles and also use as tagged proteins for various expression studies. This work is being completed with an internal collaboration with Dr. James Carroll in the Laboratory of Persistent Viral Diseases, NIAID and Dr. Lisa Olano of the Research Technologies Branch, NIAID. The long-term objective of this project is to use the improved tools and techniques to understand the basic physiology of leptospires and the mechanisms of infection and pathogenecity of L. interrogans. Together this knowledge should help accelerate the development of preventative measures against Leptospirosis.